Although antibiotics have been used successfully to prevent infection, constant use of these agents has been discouraged because some have side effects (DuPont et al., Rev. Infect. Dis. 4:533 (1982); Sack, R. B., Rev. Infect. Dis. 8:S160 (1986)). It has also been found that some microbes develop resistance to antibiotics, rendering them ineffective (McDougald, L. R., Control of Coccidiosis in Chickens: Chemotherapy. in Coccidiosis of Man and Domestic Animals, P. L. Long, ed., CRC Press, Florida, 1990, p.307). Passive immunization is an alternative method of protection from infection. Antibodies are safe, natural products that bacteria will not build up resistances against and therefore can be fed continually. No side effects were observed by Tacket et al., New Eng. J. Med. 3;18:1240 (1988) when using milk immunoglobulin concentrate as an effective prophylactic against traveler's diarrhea. Fortification of food products with specific immunoglobulins or oral vaccines containing immunoglobulins would be an innovative way to help alleviate the problems associated with antibiotics.
Egg yolk is recognized as a very good source of specific antibodies. The advantages it offers over conventional antibody production are well documented. These include the potential of producing more specific antibodies against antigens in birds and mammals (Jensenius et al., J. Immunol. Methods 46:63 ( 1981 )), low cost, convenience ( Polson et al., Immunol. Commun. 9:475 (1980)) and, what is becoming more important, compatibility with modern animal protection regulations (Gottstein, B. et al., Z. Parasitenkunde 71:273 (1985)). It has also been reported that production and maintenance of higher levels of specific antibodies is relatively easy (Orlans, E. Immunology 12:27 (1967); Rose et al., Eur. J. Immunol. 4:521 (1974)).
Hen serum IgG is transferred to its egg yolk and provides its offspring with acquired immunity. Thus, it is possible to obtain pathogen-specific egg yolk antibodies from eggs laid by hens immunized against the specific antigens. Egg yolk immunoglobulin (IgY) content of chicken eggs is about 100-150 mg/egg (Rose et al., Develop. Comp. Immunol. 5:115-20 (1981)), an amount which is remarkably higher than the antibody (IgG) content in the same volume of mammalian serum or milk. IgY differs from mammalian IgG in molecular size (168,000), isoelectric point (more acidic) (Schmizu et al., Biosci, Biotech. Biochem. 56:270-274 (1992)) and in binding ability with mammalian complement and protein A (none) (Martin et al., Can. J. Biochem. Physiol. 35:241 (1957)). IgY is also known as .gamma.-livetin and exists in egg yolk together with two other important water-soluble proteins, .alpha.-livetin (chicken serum albumin) and .beta.-livetin (.alpha.-2-glycoprotein) and various I ipoproteins (LDL and HDL) which are the major components of egg yolk (Martin et al., Can. J. Biochem. Physiol 35:241 (1957)). Therefore, the first step in the isolation of IgY is to separate the water-soluble proteins from lipoproteins. Water-soluble proteins constitute 42.4% of the total proteins in egg yolk (Osuga et al., "Egg Proteins: In Food Proteins, J. R. Whitaker and S. R. Tannenbaum eds., AVI Pub. Co., Westport, Conn. (1977)).
Many methods have been used for the isolation and purification of immunoglobulins from egg yolk (Martin et al., Can J. Biochem. Physiol. 35:241 (1957); Martin et al., Can. J. Biochem Physiol. 36:153 (1958); Jensenius et al., J. Immunol. Methods 46:63 (1981); Bade et al., J. Immunol. Methods 72:421 (1984); Polson et al., Immunol. Invest. 14:323 (1985); Hassl et al., J. Immunol. Methods 110:225 (1988)). Hatta et al. (Agric. Biol. Chem. 54:2531 (1990)) used food-grade natural gums (e.g., carrageenan) to remove yolk lipoprotein as a precipitate and to recover IgY in the water-soluble fraction from egg yolk. Kwan et al., J. Food Sci. 56:1537 (1991) used water dilution to fractionate water-soluble from water-insoluble components of egg yolk. About 15-21% of the total proteins were recovered in the supernatant of ten-fold diluted yolk, with approximately 60% recovery of IgY activity. Recently, Akita et al., (J. Food Sci. 57:629(1992)) reported that egg yolk diluted six-fold with water at pH 5.0-5.2 and incubated at 4.degree. C. for 6 hr yielded 100 mg of electrophoretically-pure IgY per egg by a combination of ultrafiltration, gel filtration and ion exchange chromatography.
Passive protection of neonatal piglets against fatal enteric coli bacillosis was achieved with powder preparations of specific antibodies against K88, K99, and 987P fimbrial adhesions of enterotoxigenic Escherichia coli (Yokoyama et al., Infect. Immunol. 60:998 (1992)). The antibody powders were obtained by spray-drying the water-soluble protein fraction of egg yolks from immunized hens after the lipid components were precipitated with an aqueous dispersion of acrylic resins (Eudragit L30D-55; Rhom Pharma).
Using the small animal model, passive protection of suckling mice against human rotavirus infection was achieved with the use of immunoglobulin from the yolks of eggs of rotavirus-immunized hens (Ebina et al., Microbiol. Immunol. 34:617 (1990)). Egg yolks separated from albumen were mixed with the same volume of distilled water. After homogenization, yolks were filtered through gauze. The precipitate fraction was found to be composed of lipoprotein, and the supernatant fraction was composed of water-soluble protein. Water-soluble protein fractions (0.123 mg IgY/mg protein) were applied to a DEAE-Sephacel ion-exchange chromatography column and eluted with 200 mM phosphate buffer, pH 8.0. Fractions of a 280 nm peak (0.562 mg IgY/mg protein) were precipitated by saturated ammonium sulphate 3 times and dialyzed with 20 mM phosphate buffer, pH 8.0. Purified IgY fractions were finally filtered through a 0.45 .mu.m membrane filter and freeze-dried.
Most of the methods mentioned, while perhaps satisfactory on a laboratory scale, are unsuited to scaling to a high volume procedure for the production of kilogram levels of food-grade antibodies. Chanutin and coworkers (Arch. Biochem. Biophys. 89:220 (1960)) observed the precipitation of plasma proteins by short-chain fatty acids at pH 4.2. Steinbuch and Audran (Arch. Biochem. Biophys. 134:279 (1969)) indicated that precipitation of the bulk of plasma proteins with caprylic (octanoic) acid can be done without adversely affecting IgG, ceruloplasmin, and part of the IgA.
Medium-chain triglycerides (MCT) are the basis of a new group of fats known as structured lipids which have advantages in clinical nutrition and the treatment of disease (Kennedy, J. P., Food Technol. 45:76 (1991)). MCT oil is a saturated fat composed of C8 (caprylic) and C10 (capric) fatty acids. It is manufactured from the fractionated medium-chain fatty acids of vegetable oils such as coconut and palm kernel oil. Medium-chain fatty acids are not incorporated into chylomicrons, therefore they are not likely to be stored in the adipose tissue of the body. Instead, they are oxidized for energy in the liver as are carbohydrates and provide a dense source of calories which the body can readily use. This makes MCT ideal for anyone with high energy requirements. In the flavor industry, MCT's are frequently used as a solvent, carrier or diluent to cut concentrated essential oils and flavors when high stability, low viscosity and blandness are desired. MCT's are also used as carriers for colors, vitamins, and pharmaceuticals. Caprylic acid is a natural component of chicken eggs, found at 0.6% in the whole egg and 0.15 % in egg yolk (Cotterill et al., Poult. Sci. 56:1927 (1977)).